Molecular cloning of the Escherichia coli miaA gene involved in the formation of delta 2-isopentenyl adenosine in tRNA

Abstract

Escherichia coli mia strains were shown to lack delta 2-isopentenylpyrophosphate transferase activity, the first step in the synthesis of the 2-methylthio derivative of 6-(delta 2-isopentenyl) adenosine (ms2i6A). A double mutant, rpsL (Smp) miaA, was streptomycin dependent. The wild-type miaA gene was cloned by selecting for lambda recombinant bacteriophage which eliminated the streptomycin-dependent phenotype and was subsequently recloned into plasmid vectors. The cloned miaA gene restored the ms2i6A modification to tRNA. The miaA gene mapped to 95 min on the E. coli map, and we propose the order mutL-miaA-hflA-purA.